Cellular interactions between antigen (Ag)-specific T cells and antigen-presenting cells (APCs) can trigger either productive immune responses or aborted immune responses. These diverse biologic outcomes are determined by the complex integration of signals that are delivered upon the ligation of multiple receptors on the membrane of T cells with their counter receptors on the membrane of APCs. Uncovering the complex integrated cellular and molecular mechanisms, that determine the fate of the interacting cells, is the long term objective of these studies. Using novel 3-D digital imaging of T-APC conjugates it was recently shown that signaling and adhesion proteins are recruited to the cell contacts and form spatially segregated Supra-Molecular Activation Clusters (SMACs). This application is highly focused on the structure and function of the c-SMAC, which is the site of TCR engagement and activation. The central premise of this proposal is that TCR associated activation events are spatially and temporally orchestrated in the newly discovered c-SMAC. To test this hypothesis we will combine the use of T cell transgenic mice and multi-dimensional imaging and will define the 4-dimensional molecular translocations and association of four key activation receptors in the c-SMAC: TCR, CD45, CD4 and CD28 during T-APC interactions. These studies will be followed by derivative experiments to determine the mechanisms of association of each of these receptors with the c-SMAC and their physiological significance. The specific aims are: Aim 1. To determine the precise temporal and 3-dimensional molecular composition of c-SMACs in cell conjugates formed between T cells from TCR trangenic mice and B-APCs. Aim 2. To study the mechanisms that cause the transient colocalization of CD45 with Ag-bound TCR and Lck in early c-SMACs and to assess the functional significance of these translocations. Aim 3. To study the mechanisms that cause the transient early association of CD4 with the engaged TCR and Class II in c-SMACs, using TCR and CD4 tailless transgenic mice. Aim 4. To study the mechanisms responsible for the association of CD28 with the engaged TCR in the cSMAC and the role of c-SMAC-associated CD28 in T cell activation. These novel studies would provide the first 4-dimensional view of T-APC interactions and, in combination with genetic and functional studies, are very likely to generate significant better understanding of the integrated events that properly regulate immune responses. This new knowledge may be useful in future designs of better immune surveillance protocols, immunosupression drugs and new vaccines.